A safe and effective vaccine for HIV is urgently needed. While significant progress has been made in developing methods to elicit HIV-specific cellular immune responses, there is presently no available method of generating broadly cross-neutralizing antibodies against primary HIV-1 isolates. Conserved regions of gp120 that are normally hidden from the host immune response but exposed following CD4 binding represent a potential target region to elicit such antibodies. This proposal describes experiments that will generate antibodies to conserved, conformation- dependent epitopes that are exposed following gp120 binding. In order to elicit antibodies against relevant regions of gp120 that are present on virion particles, HIV-1 pseudovirions (virus-like particles) will be utilized as the immunogens. Pseudovirions will incorporate primary isolate Env molecules, and will be enhanced in their incorporation and retention of gp120 through modifications of the Env backbone or through the use of selected primary isolates. Conformational changes will be induced in pseudovirion-bound gp120 through interaction with CD4 in soluble form. Fixed conformational changes will be induced through incorporation of CD4 onto the pseudovirion surface together with Env. The ability of CD4-triggered pseudovirions to generate antibodies to conserved regions of gp120 will be established following inoculation of guinea pigs and CD4-transgenic mice. Monoclonal antibody 17b will serve as a model CD4-dependent epitope antibody for use in antibody competition binding assays. Neutralizating antibodies present in guinea pig and rabbit sera will be assayed against a panel of primary isolate viruses. In order to prove the concept that antibodies against CD4-dependent epitopes can be generated by these methods, monoclonal antibodies will be developed from CD4-transgenic mice and will be screened for 17b-like activity. A novel recombinant protein representing the bridging sheet will also be employed to screen for relevant antibodies. A recombinant poxvirus capable of producing CD4- triggered pseudovirions will be generated and evaluated in a prime-boost protocol in guinea pigs. These studies will determine the ability of pseudovirion-based approaches to elicit antibodies to conserved, CD4-exposed gp120 epitopes, and will define the capacity of the elicited antibodies to neutralize primary isolates of HIV-1.